The Role of the mitotic spindle protein RHAMM in bladder premalignancy Urothelial cell carcinoma of the bladder (UCC) ranks 5th in incidence of all cancers in the developed world and has amongst the highest rate of recurrence for any solid cancer, yet there are no reliable non- invasive methods for detecting it at its earliest stages. We have identified protein biomarkers in patient urine samples that show promise for predicting UCC recurrence. One of these biomarkers, RHAMM (receptor for hyaluronic acid mediated motility, also called Hmmr), may also have oncogenic function and thereby play a role in UCC etiology. In this proposal we will determine the function of RHAMM in bladder carcinogenesis and determine whether it can serve as a marker for monitoring the efficacy of therapeutic/chemopreventive drugs. In recent studies in our laboratory, we have used a transgenic mouse model that spontaneously develops invasive UCC (UPII-SV40Tag mice) in conjunction with DNA microarray technology to identify molecular pathways involved in early UCC development, and for identifying new biomarkers for the diagnosis and prognosis of UCC. Genes that were differentially expressed between the urothelium of UPII-SV40Tag mice and their age-matched wild type (non-transgenic) littermates at 3, 6, 20, and 30 weeks of age were identified. One of these genes, RHAMM, is important for spindle formation during mitosis and is exported to the cell surface where it is involved in motogenic signaling. Preliminary data from our laboratory has also demonstrated that RHAMM expression in urine correlates with recurrence of bladder UCC in samples from a recently completed Phase II, randomized, placebo controlled chemoprevention trial (N01 CN85186). The hypothesis underlying the first aim of this proposal is that RHAMM has pro-malignant function in bladder UCC. Over expression of this protein in non-tumorigenic bladder derived cells and primary human urothelial cells (HUCs) is expected to enhance malignant characteristics, while suppression of its expression in malignant UCC cells should suppress malignancy. In the second aim we will test the hypothesis that RHAMM levels in mouse urothelium will inversely correlate with positive response to therapeutic or preventive drug treatment, thereby supporting the use of RHAMM as a marker for therapeutic efficacy. For the first Aim, we will determine the effects of shRNA-mediated knockdown of RHAMM expression in several malignant bladder UCC cell lines. Conversely, we will determine the effects of stable overexpression of RHAMM in non-tumorigenic bladder derived cells and primary urothelial cells. The malignant characteristics of the RHAMM knockdown and over expressing cells will be thoroughly assessed. In the second Aim, we will test the chemopreventive effects of ATRA at early (pre-CIS, 8 weeks), middle (CIS to T1, 16 weeks), and later stages of early UCC development (24 weeks) in UPII-SV40Tag mice. Completing these specific aims promises to advance our understanding of the process of bladder carcinogenesis and progression, both through the study of RHAMM function and through the assessment of its expression in the context of chemopreventive and therapeutic treatment. It is also expected that this study will determine whether RHAMM can be a useful marker for monitoring efficacy of chemopreventive/therapeutic drugs. PUBLIC HEALTH RELEVANCE: Urothelial cell carcinoma (UCC) of the bladder causes substantial morbidity and mortality and has the 5th highest incidence of all cancers in the developed world, with an estimated 70,980 new cases and 14,330 deaths predicted to occur in the US in 2009. Long-term study results clearly indicate that ability to intervene at early stages and monitor the success of treatment requires the definition of early markers for bladder cancer. It is therefore of vital importance to identify gene expression changes that occur during the process of bladder carcinogenesis and progression.